Anti rage

Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21^CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).

Concentrations of protein samples were determined with Roti-Quant assays (Carl Roth, Karlsruhe, Germany). 20 μg of total protein were mixed with 4× Laemmli buffer (8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 0.008% bromophenol blue, 0.25 M Tris–HCl, all from Sigma-Aldrich) and incubated for 3 min at 95 °C. Samples were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Piscataway, USA). After 2 h of blocking with TBST buffer containing 3% of bovine serum albumin (Carl Roth) at room temperature, the membranes were incubated overnight at 4 °C with specific primary antibodies, anti-CCL26 (1:100; R&D systems, Minneapolis, USA), and anti-RAGE (1:4000; Genetex, Irvine, USA). Signals from peroxidase-conjugated secondary antibodies (anti-rabbit, Cell Signaling Technology, 1:20,000) were detected using the ECL kit (ECL-Plus Western blotting Detection Reagents, Amersham Pharmacia Biotech). Protein bands on the membranes were visualized with the western blot imaging system Fusion FX (Vilber, Collégien, France) and intensities of the respective protein bands were analyzed using the ImageJ software 1.41 (NIH).